Calcium is a ubiquitous signaling ion, especially in excitable cells such as neurons. Although there are numerous receptor proteins for Ca2+, the most prominent is calmodulin (CaM). The Ca2+/CaM complex can modulate the functionality of numerous proteins in the cells including several Ser/Thr protein kinases. The Ca2v/CaM-dependent protein kinase (CaM-K) cascade is made up of CaM-kinase I (CaM-KI), CaM-kinase IV (CaM-KIV) and their upstream activating kinase CaM-kinase kinase (CaM-KK). These CaM-Ks are particularly abundant in neurons and are important in diverse cellular functions such as gene transcription, microtubule assembly, apoptosis, and synaptic plasticity. The CaM-K cascade also exhibits regulatory cross-talk with other signaling pathways such as the cAMP and MAP-kinases. The investigations proposed in this application will focus on CaM-KK. These studies will utilize purified, recombinant enzymes as well as endogenous or transfected CaM-KK mutants in cultured cells.Major components of these studies will include: 1. Investigating the regulation of CaM-KK by phosphorylation/dephosphorylation in a variety of cells. CaM-KK activity can be modulated in vitro by PKA and CaM-KI through phosphorylation of identified sites, and preliminary evidence indicates dynamic changes in cultured cells. We will extend these studies using cultured cells stimulated by agonists. Attempts will be made to identify the relevant kinases and phosphatases using selective inhibitors. 2. Web based motif programs predict that CaM-KK can interact with several scaffold or bridging proteins, and these predictions are supported by our preliminary studies. Motifs in CaM-KK required for these interactions will be determined, and the effects of agonist treatments of cultured cells on these interactions will be determined. 3. CaM-KK may be localized in the cell due to its interactions with these proteins. We are particularly interested in the dynamic localization of CaM-KK in growth cones, where it may modulate neurite extension, and the cell nucleus, where it regulates gene transcription through CaM-KIV.These studies will further our understanding of the functions of CaM-KK in cellular Ca2+ signaling and their roles in neurotransmitter/hormone responses, neuronal development and cell death, and synaptic plasticity.